Compositions for regenerating tissue that has deteriorated, and methods for using such compositions

ABSTRACT

A composition for promoting regeneration of tissue which has degenerated in a subject as a result of a disease or disorder and a method of using the composition is provided. The composition comprises a biodegradable acellular matrix, and passaged autologous fibroblasts substantially free of immunogenic proteins, e.g., culture medium serum-derived proteins, integrated within the matrix. The method of using the composition to promote regeneration of tissue involves placing the composition on a site of degenerated tissue in a subject so that the composition promotes tissue regeneration at the site. The composition and the method of its use have applications promoting regeneration of tissue (i) that has degenerated as a result of numerous diseases or disorders or (ii) that has a defect, including, but not limited to, defects of the oral mucosa, trauma to the oral mucosa (e.g., extraction of a tooth), periodontal disease, diabetes, cutaneous ulcers, venous stasis, scars of the skin, or wrinkles of the skin. Also provided is an injectable composition comprising any type of collagen and passaged autologous fibroblasts substantially free of immunogenic proteins, e.g., culture medium serum-derived proteins, for correcting defects in skin, such as wrinkles or scars, and for augmenting tissue in the subject, particularly facial tissue.

[0001] This application is a continuation-in part and claims priority ofU.S. application Ser. No. 09/316,245, filed May 21, 1999, which is acontinuation-in-part and claims priority of U.S. application Ser. No.09/083,618, filed May 22, 1998. U.S. application Ser. Nos. 09/316,245and 09/083,618 are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

[0002] The present invention concerns the regeneration of tissues in asubject that have degenerated as a result of a disease or disorder inthe subject. More particularly, the present invention concerns novelcompositions for use in surgical and nonsurgical techniques that promoteregeneration of tissue whose mass has been diminished due to a diseaseor disorder in a subject, correct defects in the skin of subjects, oraugment tissue in subjects. Also disclosed is the use of a novelcomposition in conjunction with a biodegradable acellular matrix forameliorating defects in the tissues, and methods for using the novelcomposition.

BACKGROUND OF THE INVENTION

[0003] “Periodontal disease” is the term commonly used to describeinflammatory disease of the periodontium, i.e., the tissue surroundingand securing teeth to the jawbone. The condition is characterized byinflammatory and degenerative processes that develop at the gingivalmargin (gingivitis) and lead to a progressive breakdown and resorptionof the periodontal ligament and bone (periodontitis), oftentimesresulting in severe diminution of the periodontium. Periodontal diseaseis the leading cause of tooth loss in adults after middle age.[Anderson's Pathology, p. 2000, John M. Kissane ed., 9th ed. (1992)].

[0004] Periodontal disease results from the accumulation of bacterialplaque in the gap between the gingiva and the tooth. While anaerobicbacteria are the primary etiologic agents, the destructive process isbelieved to be mediated in large part by immunologic reactions of thehost. As the disease progresses, a periodontal pocket is establishedbelow the gingival margin, thus prolonging and promoting theinflammatory process. Successive inflammatory reactions result in theprogressive erosion of the tooth-supporting tissues, i.e., thecollagenous fibers making up the periodontal ligament and the bonepocket in which the tooth sits. [Reviewed in Anderson's Pathology, pp.1999-2000, John M. Kissane ed., 9th ed. (1992); Shafer et al., ATextbook of Oral Pathology, 4th ed. (1983)].

[0005] Periodontal disease can be diagnosed by checking the gingiva forinflammation, probing the depths of periodontal pockets, checkingclinical attachment level, and assessing bone loss by means ofautoradiography. [Jeffcoat, M. K., et al., J. Am. Dent. Assoc.,128:713-724 (1997)].

[0006] A number of techniques, both surgical and nonsurgical, have beendeveloped to treat periodontal disease. Particularly with respect tosevere periodontitis, none of the currently available treatments arewholly satisfactory.

[0007] For relatively mild cases of periodontitis, practitioners havetraditionally employed nonsurgical mechanical debridement (i.e. scalingand root planing) to remove the bacterial plaque whose accumulationperpetuates the disease, thereby reducing inflammation. Mechanicaldebridement can be accomplished using manual, sonic or ultrasonicinstruments. Scaling and root planing have been shown to decreasegingival inflammation, decrease probe depth, and promote maintenance ofclinical attachment level. However, without resorting to surgicalprocedures, access to root surfaces and bony defects is restricted, andonly limited debridement is possible. [Jeffcoat, M. K., et al., J. Am.Dent. Assoc. 128:713-724 (1997)].

[0008] As a result, nonsurgical scaling and root planing is insufficientto treat more severe cases of periodontitis, and it is necessary toresort to more aggressive surgical techniques. Surgical techniquescomprise reflecting the gingival tissues to provide access to rootsurfaces and bone defects, in order that mechanical debridement may beaccomplished directly. Following debridement, the gingival tissue issutured back in position. Currently available surgical approaches entailsubstantial patient discomfort and fail to consistently providesatisfactory outcome.

[0009] There are a number of non-surgical, non-mechanical approaches totreating periodontal disease, including supragingival and subgingivalirrigation and the application of chemical and antimicrobial agents. Yetnone of these approaches have achieved more than marginal success[Jeffcoat, M. K., et al., J. Am. Dent. Assoc., 128:713-724 (1997)]. Inparticular, there are a number of deleterious side effects associatedwith the use of antibiotics, along with risks such as drug sensitivityand the emergence of antibiotic-resistant pathogens.

[0010] Another approach to combating destruction of the periodontaltissue has focused on inhibiting the matrix metalloproteases responsiblefor this destruction. Tetracyclines in particular have shown promise asinhibitors of extracellular collagenases, but cause the same sideeffects associated with antibiotics in general. Modified forms oftetracycline have been developed which are non-antimicrobial and retaintheir ability to inhibit collagenases, but these chemically modifiedtetracyclines are not commercially available. [Ciancio, G. C. et al., J.Am. Dent. Assoc. 123:34-43 (1992)].

[0011] Because cell proliferation, cell migration and matrix synthesisare prerequisites for periodontal regeneration, some researchers haveattempted to use tissue growth factors, for example insulin-like growthfactor, platelet-derived growth factor, and transforming growth factorto promote periodontal regeneration.

[0012] In summary, none of the nonmechanical approaches to treatingperiodontitis have been able to offer more than modest, short termenhancement of traditional mechanical debridement. As noted in a 1997review of techniques used in treating periodontal disease, “scaling androot planning accompanied by oral hygiene procedures remains the firstmode of treatment for adult periodontitis.” [Jeffcoat, M. K. et al., J.Am. Dent. Assoc., 128:713-724 (1997)].

[0013] A great deal of research has been directed to methods ofregenerating periodontal tissue lost as a consequence of periodontaldisease, but as yet no wholly satisfactory method is available. For themost part, efforts have focused on surgical approaches that fill thedefects with a variety of materials (bone grafting) or use guided tissueregeneration.

[0014] Bone grafting techniques involve the use of natural bone orsynthetic bone materials. Natural bone grafts are typically eitherautografts (grafts transferred from one position in the body of apatient to another position in the body of the same patient) orallografts (grafts transferred from one person to another). Cliniciansusing natural bone grafts have had limited success in inducing new bonegrowth. Problems associated with the use of autografts include the needfor a second surgical site and, in some cases, fresh grafts may beassociated with root resorption. [Jeffcoat, M. K. et al., J. Am. Dent.Assoc. 128:713-724 (1997)].

[0015] Freeze-dried, demineralized bone has been used as an allograftand shown to promote bone formation. However, the predictability and theamount of bone fill achieved varies. [Jeffcoat, M. K. et al., J. Am.Dent. Assoc., 128:713-724 (1997)]. Since allografts are transferred fromone person to another, the potential exists that viruses or otherpathogens might be transferred to the patient.

[0016] Synthetic bone materials which have been investigated includeplaster, calcium carbonates, and ceramics such as hydroxyapatite.Clinical trials have demonstrated that the use of synthetic grafts hasresulted in improvements in probing depth and attachment level.Histologic findings, however, indicate that, in general, syntheticgrafts act primarily as space fillers, with little if any regeneration.[Jeffcoat, M. K. et al., J. Am. Dent. Assoc., 128:713-724 (1997)].

[0017] Guided tissue regeneration is a surgical approach based onplacing a membrane barrier under a soft tissue flap above the area ofbone loss to enhance wound healing potential. [Ciancio, G. C. et al., J.Am. Dent. Assoc., 123:34-43 (1992)]. Investigators have studied bothresorbable and nonresorbable membranes. A significant disadvantage ofusing a nonresorbable membrane is the requirement of a second surgicalprocedure after approximately six weeks to remove the membrane.Furthermore, in about 40%-50% of the cases, such membranes becomeinfected in the patient. While less evidence is available for resorbablemembranes than for nonresorbable membranes, improvements in clinicalattachment levels have been shown for both types of membranes comparedwith debridement alone. Most favorable results are reported for Class IIfurcations in the mandible and for intrabony defects. Less favorableresults have been reported in maxillary molar and Class III (through andthrough) furcation defects [Jeffcoat, M. K. et al., J. Am. Dent. Assoc.,128:731-724 (1997)].

[0018] In summary, none of the currently available treatments forperiodontal disease is wholly satisfactory, particularly with regards toregenerating periodontal tissue lost as a result of periodontitis.

[0019] The oral mucosa is the tissue lining the oral cavity. There are anumber of conditions that can result in defects in the oral mucosa, forexample trauma, dermatoses, recurrent aphthous stomatitis, andinfections. [Flint, S., The Practitioner 235:56-63 (1991)]. There iscurrently no wholly satisfactory means of correcting these defects.

[0020] Furthermore, there have been efforts to develop and usecompositions and methods to correct defects in skin, such as scars andwrinkles, or to augment the tissue of a subject in order to improve theappearance of the skin, particularly facial skin. The principal methodemployed to correct such defects involves injecting a filler compositioninto the dermal layer of the skin proximate to the defect or desiredtissue augmentation. Examples of non-biological filler compositions usedin these roles include mineral oil, paraffin, silicone fluid, autologousfat, gelatin powder mixes, polymethylmethacrylate microspheres,cross-linked polydimethylsiloxane, ““TEFLON”” paste, reconstitutedbovine collage, and autologous human collagen.

[0021] However, the use of these compositions comprises inherentlimitations. For example, the use of mineral oil, paraffin and similaroils and waxes has resulted in complications such as local chronicedema, lymphadenopathy, scarring and ulcerations (Devore et al.,Effectiveness of injectable filler materials for smoothing wrinkle linesand depressed scars. Medical Progress Through Technology 20:243-250(1994 which is hereby incorporated by reference in its entirety).

[0022] The use of reconstituted bovine collagen to correct defects oraugment tissue also possesses inherent limitations. For example, it hasbeen reported that reconstituted bovine collage is only moderatelyeffective, and is associated with infrequent, but controversial, adversereactions. In addition, it is rapidly broken down and resorbed in vivo,providing only a temporary correction of a skin defect or augmentation.More importantly, reconstituted bovine collagen may elicit an immuneresponse in the subject. Id.

[0023] As explained above, gelatin matrix implant such as that soldunder the mark “FRIBEL”, is a composite material of porcine gelatinpowder and o-aminocaproic acid which are dispersed in 0.9% (by volume)sodium chloride solution and an aliquot of the recipient's plasma mixedin a 1:1 ratio, is also used to correct skin defects and augment tissue.However, this material also possess inherent limitations. Specificallygelatin matrix does not appear to have applications in the treatment ofwrinkle lines. Moreover, since a large bore needle (27 gauge or greater)is used to inject the gelatin matrix into the subject's skin, treatmentwith gelatin results in greater discomfort and pain to the subject asopposed to the injection of other fillers. Id.

[0024] In addition, the use of autologous fat injections to correct askin defect or augment tissue in a subject, while eliminating thepotential of eliciting an immune response, also possesses disadvantages.More specifically, prior to its injection, fat must be processed byskilled clinicians in aseptic conditions to maintain sterility. Inaddition, the injections are not dermal but are subcutaneous orsubdermal. Also, a very large bore needle (as large as 16 gauge) isneeded to inject the fat into a subject, resulting in great pain,moderate bruising, and formation of visible puncture holes. Moreover,fat injections are subject to rapid resorption, and must be repeated inorder to maintain skin augmentation or defect correction.

[0025] The use of autologous, injectable dermal collagen to correctdefects or augment tissue has also met with limited success. Forexample, if large concentrations of collagen are injected, a 27 gaugeneedle or larger is used, resulting in the infliction of pain on thesubject. Furthermore, serial injections are required in order tocompensate for the gradual resorption of autologous collagen.

[0026] Hence, what is needed is an efficient non-surgical compositionthat promotes the regeneration of tissues of the gums or the palate andbone that have degenerated as a result of periodontal disease or trauma.

[0027] Moreover, what is needed is a composition and method forpromoting regeneration of tissue that does not elicit an immune responsein the subject at the site of desired tissue regeneration.

[0028] What is also needed is a composition that can be used innon-surgical methods to correct defects in skin, such as scars orwrinkles, and augment tissue in a subject, particularly facial tissue,which is not rapidly resorbed by the body so that additional injectionsare required.

[0029] U.S. Pat. No. 5,591,444, U.S. Pat. No. 5,660,850, and U.S. Pat.No. 5,665,372 are incorporated herein by reference in their entirety.

SUMMARY OF THE INVENTION

[0030] The present invention provides a composition for promotingregeneration of tissue in a subject that has degenerated as a result ofa disease or disorder, and a method of using the composition that doesnot suffer from the shortcomings of other methods described above. Thepresent invention is based on the inventors' discovery of the successfuluse of autologous fibroblasts, with and without various forms of matrix,filler or carrier material, to regenerate tissue in a subject, correctskin defects in the subject, or augment tissue in the subject.Fibroblasts are connective-tissue cells involved in tissue repair. Whena tissue is injured, nearby fibroblasts migrate into the wound,proliferate, and produce large amounts of collagenous matrix, whichhelps to isolate and repair the damaged tissue. [Alberts et al.,Molecular Biology of the Cell, p. 987, 2nd ed., (1992)].

[0031] Broadly, the present invention extends to a method forregenerating a subject's tissue (a) that has degenerated as a result ofa disease or disorder or (b) that has a defect, comprising the steps ofproviding a pharmaceutical composition comprising autologous, passagedfibroblasts, identifying a site of tissue degeneration, and injecting aneffective amount of the composition into tissue at the site of tissuedegeneration so that the tissue is augmented and regeneration of tissueis promoted.

[0032] Injection of a pharmaceutical composition of the presentinvention can be into tissues of the subject comprising the periodontalpocket and/or the periodontal tissue adjacent to the area ofdegeneration or into tissue subadjacent to a defect in the oral mucosa,or into the tissue of the palate of a human subject, in order promoteregeneration of tissue in the oral mucosa, the gingiva, or the palate.Typical defects in the oral mucosa or palate that can be corrected withthis embodiment of the present invention include those caused by trauma,dermatoses, recurrent aphthous stomatitis, and infections, or a diseaseor disorder. Moreover, the present invention can be used to correctdefects in the skin, such as scars, wrinkles, laugh lines, rhytids,stretch marks, depressed scars, cutaneous depressions of non-traumaticorigin, acne scarring, or subcutaneous atrophy from acne, trauma,congenital malformation, or aging. Moreover, the invention can be usedto treat defects such a hypoplasia of the lips, or labial folds. Inaddition, the composition can be used to repair a defect, disorder ordisease of bone, e.g., bones such as, for example, facial bonesincluding orbits, mandibles, maxillae, zygomatic bones, crania, andnasal bones. Bone diseases, disorders, or defects, include, for example,tooth extraction-related bone defects or those due to periodontaldisease.

[0033] A disease or disorder which results in tissue degeneration in asubject that can be treated with the present invention includes, but isnot limited to, defects of the oral mucosa, periodontal disease, traumato the oral mucosa (e.g., extraction of a tooth), diabetes, cutaneousulcers, or venous stasis. Moreover, periodontal disease can includeperiodontal degeneration, gingivitis, or a non-healing wound of thepalatal mucosa or the gingival mucosa.

[0034] The present invention further extends to a method of forming acomposition comprising autologous, passaged fibroblasts which aresubstantially free of immunogenic proteins, such as culture mediumserum-derived proteins, and are histocompatible with a subject. Thismethod comprises the steps of collecting a biopsy of dermis from asubject, isolating the autologous fibroblasts contained in the biopsyfrom extracellular matrix and other cells contained in the biopsy,culturing the autologous fibroblasts in a culture medium that permitsexpansion of the autologous fibroblasts, incubating the autologousfibroblasts in a protein free medium for at least about 2 hours betweenabout 30° C. and about 37.5° C., and exposing the incubated autologousfibroblasts to a proteolytic enzyme so as to suspend the fibroblasts. Anexample of a culture medium that permits expansion of autologousfibroblasts comprises between 0.0% and about 20% serum, wherein theserum can be either human or non-human. Also, the biopsy from dermis cancomprise tissue from the gums, palate or skin of the subject. Henceautologous fibroblasts from the gums, plate or skin have applications inthe present invention.

[0035] In another embodiment, the passaged autologous fibroblasts can beadded to a pharmaceutically acceptable carrier to form a pharmaceuticalcomposition. In producing a pharmaceutical composition of the presentinvention, immunogenic proteins, e.g., culture medium serum-derivedproteins, are removed from the autologous fibroblasts, thereby avoidingan immunological reaction in a subject when such cells are reintroducedto the subject proximate to the site of tissue degeneration or defect.

[0036] In addition, the present invention extends to a device fordelivering a pharmaceutical composition of the present invention to asite proximate to the site of tissue degeneration or defect in asubject, wherein the device comprises a hypodermic syringe having asyringe chamber, a piston disposed therein, an orifice communicatingwith the chamber, a pharmaceutical composition comprising autologouspassaged fibroblasts and a pharmaceutically acceptable carrier, suchthat the pharmaceutical composition is disposed in the chamber, and ahypodermic needle is fixed to the orifice.

[0037] Tissues which have suffered degeneration or have a defect thatcan be treated with a device of the present invention include the oralmucosa, the gingival mucosa, and the palatal mucosa. Moreover, diseasesor disorders which can be treated with this device include periodontaldegeneration, gingivitis, or a non-healing wound of the palatal mucosaor the gingival mucosa. Other defects that can be treated with thisinvention include those listed above.

[0038] The present invention further extends to a composition forrepairing tissue that has degenerated in a subject as result of adisease, disorder or defect in the subject, wherein the compositioncomprises a biodegradable acellular matrix, and autologous passagedfibroblasts (derived, for example, from the gums, palate, or skin of thesubject) and is substantially free of immunogenic proteins, e.g.,culture medium xenogeneic (e.g., fetal bovine) serum-derived proteins,wherein the autologous fibroblasts are integrated into the biocompatiblebiodegradable acellular matrix. In an embodiment of the presentinvention, the biocompatible biodegradable acellular matrix comprisesexogenous proteins, such as any type of collagen. In addition, thebiodegradable acellular matrix can be comprised of any type of collagenand glycosaminoglycans (GAG) cross-linked with, for example,glutaraldehyde, or any type of collagen.

[0039] In yet another example, the biodegradable acellular matrixcomprises one or more of gelatin, polyglycolic acid, cat gut,demineralized bone, or hydroxyapatite. Other appropriate matricesconsist of bone from which substantially all (e.g., at least 80%, atleast 90%, at least 95%, at least 99%, or even 100% by weight) organicmaterial has been removed (referred to herein as “anorganic bone”); suchmatrices can, optionally, include exogenous collagen in various amounts(e.g., about 1%, about 2%, about 5%, about 10%, or about 20% by dryweight).

[0040] Also, diseases, disorders or defects resulting in degeneration oftissue in a subject which can be treated with the present invention,comprise defects of the oral mucosa, trauma (e.g., extraction of atooth) to the oral mucosa or oral bones such as the maxillary ormandibular bones, periodontal disease, diabetes, cutaneous ulcers, orvenous stasis. In addition, examples of periodontal disease which resultin tissue degeneration include, but are not limited to, periodontaldegeneration, gingivitis, or non-healing wounds of the palatal mucosa orgingival mucosa, or bone degeneration. Other defects that can be treatedwith this invention include skin defects, such as scars, wrinkles, laughlines, stretch marks, depressed scars, cutaneous depressions ofnon-traumatic origin, acne scarring, or subcutaneous atrophy from acne,trauma, congenital malformation, or aging. Moreover, the invention canbe used to treat defects such a hypoplasia of the lips, labial folds, orbone defects, e.g., defects of bones such as, for example, facial bonesincluding orbits, mandibles, maxillae, zygomatic bones, crania, andnasal bones.

[0041] Also encompassed by the invention is a method for making acomposition for the repair of tissue that has degenerated in a subjectas result of a disease, disorder, or defect in the subject. The methodcomprises: providing a suspension of autologous, passaged fibroblasts;providing a biodegradable acellular matrix;

[0042] incubating the suspension of autologous passaged fibroblasts withthe biodegradable acellular matrix such that the autologous passagedfibroblasts integrate within the biodegradable acellular matrix; andremoving substantially all culture medium serum-derived proteins fromsaid biodegradable acellular matrix and said integrated fibroblasts toform a composition for promoting the repair of tissue. Sufficientautologous, passaged fibroblasts integrate within the biodegradableacellular matrix to substantially fill the space on and within thebiodegradable acellular matrix available for cells. As used herein,“substantially” fill with passaged autologous fibroblasts means to fillto a level sufficient to prevent an amount of cell proliferation thatdegrades a collagen matrix to a practically deleterious level, as aperson skilled in the art can readily determine for a particularembodiment.

[0043] The biodegradable acellular matrix used in this method cancontain exogenous protein such as, for example, any type of collagen,e.g., any type of collagen and glycosaminoglycans, cross-linked with,for example, glutaraldehyde. The biodegradable acellular matrix cancontain one or more of the following substances: gelatin, polyglycolicacid, cat gut, demineralized bone, hydroxyapatite, gelatin, polyglycolicacid, cat gut, or anorganic bone with or without any of range ofconcentrations of exogenous collagen (see below).

[0044] The disease, disorder, or defect to be treated can be a defect ofan oral mucosa, trauma to an oral mucosa, periodontal disease, diabetes,a cutaneous ulcer, venous stasis, a scar of skin, or a wrinkle of skin.Alternatively, the disease or disorder can be periodontal disease, andthe periodontal disease can be periodontal degeneration, gingivitis, ora non-healing wound of a palatal mucosa or a gingival mucosa.

[0045] In this method the step of providing a suspension of autologous,passaged fibroblasts can involve: collecting a biopsy of dermis orpalate of the subject; separating dermal autologous fibroblasts from thebiopsy; culturing the dermal autologous fibroblasts in a culture mediumcontaining (a) between 0.0% and about 20% human or non-human serum and(b) a reagent that prevents the growth of mycoplasma; and exposing theincubated dermal autologous fibroblasts to a proteolytic enzyme so as tosuspend fibroblasts. The step of collecting a biopsy of dermis caninvolve collecting a biopsy from gums, palate or skin of the subject.The reagent can contain tylosin and, optionally, one or more of thefollowing compounds: gentamicin, ciprofloxacine, alatrofloxacine,azithromycin, or tetracycline.

[0046] The present invention further extends to a method of using acomposition for promoting regeneration of tissue, wherein the methodcomprises providing passaged autologous fibroblasts integrated into abiodegradable acellular matrix, identifying a site (i) of tissuedegeneration due to a disease or disorder in the subject or (ii) adefect in the tissue of the subject, and placing the composition on thesite so that the tissue is repaired. Autologous passaged fibroblastsused herein can comprise fibroblasts from the gums, palate or skin ofthe subject.

[0047] Diseases, disorders, or defects which can be treated with thismethod include, but are not limited to, defects of the oral mucosa,trauma to the oral mucosa (e.g., extraction of a tooth), periodontaldisease, diabetes, cutaneous ulcers, or venous stasis. Examples ofperiodontal disease that can be treated with the present inventioncomprise periodontal degeneration, gingivitis, or a non-healing wound ofthe palatal mucosa or the gingival mucosa. Moreover, defects (e.g., skindefects such as scars or wrinkles) can be treated with the compositionof the present invention. In a preferred embodiment, such defects aretreated with a composition comprising fibroblasts from the palate. Anyof the above listed diseases, disorders, or defects can also be treatedby these methods.

[0048] Furthermore, biodegradable acellular matrices having applicationsin the present invention may comprise exogenous proteins. Examples ofsuch matrices include matrices comprising any type of collagen, or anytype of collagen and glycosaminoglycans (GAG) cross-linked with, forexample, glutaraldehyde.

[0049] Other examples of biodegradable acellular matrices havingapplications in the present invention include one or more of gelatin,polyglycolic acid, cat gut, demineralized bone (e.g., demineralizedhuman bone), or hydroxyapatite. Other appropriate matrices consist ofbone from which substantially all (e.g., at least 80%, at least 90%, atleast 95%, at least 99%, or even 100% by weight) organic material hasbeen removed (referred to herein as “anorganic bone”); such matricescan, optionally, include exogenous collagen, in various amounts (e.g.,about 1%, about 2%, about 5%, about 10%, or about 20% by dry weight).

[0050] The present invention further extends to an injectablecomposition for correcting a defect in skin of a subject, or augmentingtissue of a subject, said injectable composition comprising passaged,autologous fibroblasts substantially free of immunogenic proteins, e.g.,culture medium serum-derived proteins, and a biodegradable, acellularinjectable filler material. Passaged autologous fibroblasts havingapplications in an injectable composition of the present invention arefrom gums, palate or skin of the subject.

[0051] Furthermore, the present invention extends to an injectablecomposition as described above, wherein the biodegradable, acellularinjectable filler material comprises endogenous proteins. In particular,the acellular injectable filler material of an injectable composition ofthe present invention comprises an injectable dispersion of autologouscollagen fibers having a concentration in the composition of at least 24mg/ml of composition.

[0052] In addition, the present invention extends to an injectablecomposition as described above, wherein the biodegradable acellularinjectable filler material comprises exogenous proteins, such as anytype of collagen. An example of an exogenous collagen havingapplications in an injectable composition of the present invention isreconstituted bovine collagen fibers cross-linked with glutaraldehyde.

[0053] Furthermore, the filler material can comprise any type ofsolubilized gelatin either alone, or in combination with othermaterials. In a particular example, the filler material comprisesporcine gelatin powder and o-aminocaproic acid dispersed in sodiumchloride solution and an aliquot of plasma from the subject to beinjected with the composition. Preferably the ratio of sodium chlorideto serum is 1:1 by volume. Other examples of materials havingapplications in the present invention as biodegradable, acellularinjectable filler material include, but are not limited to polyglycolicacid or cat gut.

[0054] The present invention further extends to a method for correctinga defect in skin of a subject, or augmenting tissue of a subject,wherein the method comprises injecting an effective amount of aninjectable composition comprising autologous passaged fibroblastssubstantially free of immunogenic proteins (e.g., culture mediumserum-derived proteins) and a biodegradable, acellular injectable fillermaterial, into the skin of the subject at the site of the skin defect ordesired tissue augmentation, so that regeneration of tissue at the siteis promoted at the site.

[0055] Moreover, the present invention extends to a method forcorrecting a defect in skin of a subject, or augmenting tissue of asubject, the method comprising the steps of injecting autologousfibroblasts substantially free of immunogenic proteins, e.g., culturemedium serum-derived proteins, into the subject at a site of a skindefect or desired tissue augmentation, and subsequently injecting abiodegradable, acellular injectable filler material into the site. In aparticular embodiment of this method of the present invention, theduration between injecting the autologous fibroblasts into the subjectand injecting the biodegradable acellular injectable filler into thesubject is about two weeks.

[0056] Autologous fibroblasts having applications in methods of thepresent invention for correcting a defect in skin of a subject, oraugmenting tissue of a subject can be obtained from the gums, palate ofskin of the subject.

[0057] The present invention further extends to a method for correctinga defect in skin of a subject, or augmenting tissue of the subject, asdescribed above, wherein the biodegradable, acellular injectable fillermaterial comprises endogenous proteins. For example, the biodegradableacellular injectable filler material can comprise an injectabledispersion of autologous collagen fibers, preferably at a concentrationof at least 24 mg of autologous fibers per ml of composition.

[0058] Furthermore, the present invention extends to a method forcorrecting a defect in skin or other tissues of a subject (such as thosedescribed above), or augmenting tissue of the subject, also as describedabove, wherein the biodegradable, acellular filler material of thecomposition comprises exogenous proteins such as, for example, any typeof collagen. An example of collagen having applications in a method ofthe present invention comprises reconstituted bovine collagen fiberscross-linked with, for example, glutaraldehyde.

[0059] Other examples of biodegradable, acellular injectable fillermaterial for use in a method for correcting a defect in skin or asubject, or augmenting tissue of the subject include, but are notlimited to solubilized gelatin, polyglycolic acid, or cat gut sutures.More specifically, an example of acellular injectable filler materialhaving applications in the present invention comprises porcine gelatinpowder and aminocaproic acid dispersed in sodium chloride solution, andan aliquot of plasma from the subject. Preferably, the ratio of sodiumchloride solution to the aliquot of serum is 1:1 by volume. Furthermore,the sodium chloride solution comprises 0.9% sodium chloride by volume.

[0060] In addition, the present invention extends to a method forcorrecting a defect in skin of a subject, or augmenting tissue of asubject, as described above, wherein the ratio of autologous fibroblastssubstantially free of immunogenic proteins (e.g., culture mediumserum-derived proteins) biodegradable, acellular injectable fillermaterial is approximately 1:1 by volume.

[0061] Accordingly, it is an object of the present invention to providea composition for augmenting tissue, or promoting regeneration of tissuesuch as the oral mucosa, the gingival mucosa, or the palatal mucosa orskin, which has degenerated as a result of a disease or disorder.Examples of such disorders include periodontal disease, trauma,dermatoses, recurrent aphthous stomatitis, infections, scars, orwrinkles and the others listed above.

[0062] It is another object of the present invention to provide acomposition for augmenting tissue, or promoting regeneration of tissue,wherein the composition is histocompatible with a subject, therebyavoiding elicitation of an immune response and inflammation in thetissues of the subject near the site of degeneration of tissue.

[0063] It is yet another object of the present invention to providemethod of promoting tissue regeneration that does not require surgery.

[0064] It is yet still another object of the present invention topromote regeneration of tissue in a subject without the use ofantibiotics in the subject, and hence prevent the emergence ofantibiotic resistant pathogens and deleterious side effects associatedwith antibiotics in the subject.

[0065] It is another object to provide an injectable composition forcorrecting defects in skin, such as scars or wrinkles, or for augmentingtissue in a subject, particularly facial tissue (such as lips), whichincludes passaged autologous fibroblasts that can withstand resorptionso that subsequent injections are not needed, and to prevent theelicitation of an immune response in the subject.

[0066] It is yet another object of the present invention to providemethods for correcting defects in skin, such as scars or wrinkles, orfor augmenting tissue in a subject, that employs the injectablecomposition set forth above, that inflicts limited pain on the subject,and does not elicit an immune response in the subject, and is notrapidly resorbed.

[0067] Other objects and advantages will become apparent to thoseskilled in the art from a review of the ensuing description.

DETAILED DESCRIPTION OF THE INVENTION

[0068] The present invention is based, in part, on the recognition thatliving cells normally present in tissue that has degenerated,particularly fibroblasts, are the ideal material to augment the volumeof tissue in order to promote regeneration of tissue. Hence the presentinvention ameliorates and reverses the degenerative effects of a diseaseor disorder which results in tissue degeneration.

[0069] Additionally, the present invention is based on the recognitionthat an ideal composition with which to augment the oral mucosasubadjacent to a defect, to treat defects in the palate or gingiva, orto promote regeneration of tissue that has degenerated as a result ofperiodontal disease, would comprise living cells normally present insuch tissues, particularly fibroblasts.

[0070] Moreover, the present invention is based on the recognition thatan abundant supply of autologous cells of the desired type can beobtained by culturing a biopsy specimen taken from the skin, palate orgums of a subject several weeks prior to treating the tissuedegenerating disease, disorder, or defect. The invention is furtherbased on the recognition that, after such a tissue culture expansion,the autologous cells will contain a significant quantity of immunogenicproteins, e.g., proteins derived from xenogeneic (e.g., bovine, horse,goat, or sheep) serum used to supplement the medium used for tissueculture, but that the immunogenic proteins can be removed, prior totreatment of the subject.

[0071] The term “biodegradable” as used herein denotes a compositionthat is not biologically harmful and can be chemically degraded ordecomposed by natural effectors (e.g., weather, soil bacteria, plants,animals).

[0072] The term “autologous” as used herein refers to cells removed froma donor and administered to a recipient, wherein the donor and recipientare the same individual.

[0073] The term “effective amount” as used herein refers to theinjection of an amount of pharmaceutical composition of the presentinvention to promote tissue regeneration in of tissue that hasdegenerated in a subject.

[0074] As used herein, a composition or cells (e.g., autologous passagedfibroblasts) that are “substantially free of culture mediumserum-derived proteins” are a composition or cells in which the fluidsurrounding the composition or cells or incorporated into the body ofbiodegradable acellular matrices that are components of suchcompositions contains less than 0.1% (e.g., less than 0.02, 0.04, 0.008,or 0.0016%) of the xenogeneic serum contained in the tissue culturemedium in which the composition or cells were last cultured.

EXAMPLE I Administration of a Suspension of Autologous Fibroblasts toPromote Tissue Regeneration and Correct Defects in Tissues Method ofObtaining an Injectable Cell Pharmaceutical Composition

[0075] As disclosed above, one embodiment of the present inventioncomprises a method for regenerating tissue that has been damaged in asubject as a result of a disease or disorder in the subject, wherein themethod comprises providing a pharmaceutical composition comprising ofautologous, passaged fibroblasts substantially free of immunogenicproteins (e.g., culture medium serum-derived proteins), identifying asite of tissue degeneration or a defect in tissue, injecting aneffective amount of the pharmaceutical composition into the tissue atthe site of the tissue degeneration or defect so that the tissue isaugmented, and the growth of tissue is promoted at the site ofdegenerated tissue.

[0076] A disease or disorder which promotes tissue degeneration in asubject, and can be treated with this aspect of the present inventionincludes, but is not limited to, defects of the oral mucosa, trauma tothe oral mucosa, periodontal disease, diabetes, cutaneous ulcers, orvenous stasis. Furthermore, examples of periodontal disease which can betreated with this aspect of the present invention include periodontaldegeneration, gingivitis, or a non-healing wound of the palatal mucosaor the gingival mucosa. In addition, defects in skin and other tissues(see above) can be treated with the present invention.

[0077] The invention can be practiced by injecting any undifferentiatedmesenchymal cell that can be expanded in culture. In a preferredembodiment, dermal fibroblasts are injected because they can be readilyobtained and expanded, and because they are a cell type normally presentbeneath the gingival mucosa or palatal mucosa. Fibroblasts taken from abiopsy of the gums, palate, or skin of the subject can be used in thepresent invention.

[0078] In providing a composition of the present invention, a dermalfibroblast culture is initiated from a 1 to 5 mm full thickness biopsyspecimen of the gums, palate or skin of a subject suffering from tissuedegeneration. Because of the phenomenon of allograft rejection, which iswell known to transplantation surgeons and immunologists, it isessential that the cultured fibroblasts be histocompatible with thehost. Histocompatibility can be ensured by obtaining a biopsy of thesubject to be treated and culturing the fibroblasts from this specimen.

[0079] Before the initiation of the culture, the biopsy is washedrepeatedly with antibiotic and antifungal agents. An exemplary “washmedium” can contain tissue culture medium such as Dulbecco's ModifiedEagle's Medium (DMEM) and all or some of the following antibiotics:gentamicin, amphotericin B (Fungizone), and tylosin (manufactured byGibco-BRL and sold by Life Technologies, Rockville, Md., as “Anti-PPLO”,PPLO being an acronym for “pleuropneumonia-like organism”, now known as“mycoplasma”). Gentamicin can be used at a concentration of 0.1-5.0(e.g., about 0.5) mg/ml. Amphotericin B can be used at a concentrationof 0.0005-0.0125 (e.g., about 0.0025) mg/ml. Tylosin can be used at aconcentration of 0.012-1.2 (e.g., 0.12) mg/ml. The specimen of dermis isthen separated into small pieces. The pieces of the specimen areindividually placed onto the dry surface of a tissue culture flask andallowed to attach for between about 5 and about 10 minutes, before asmall amount of medium is slowly added, taking care not to displace theattached tissue fragments. After about 48 hours of incubation, the flaskis fed with additional medium. When a T-25 flask is used to start theculture, the initial amount of medium is about 1.5-2.0 ml. Theestablishment of a cell line from the biopsy specimen ordinarily takesbetween about 2 and 3 weeks, at which time the cells can be removed fromthe initial culture vessel for expansion.

[0080] During the early stages of the culture, it is desirable that thetissue fragments remain attached to the culture vessel bottom; fragmentsthat detach should be reimplanted into new vessels. The fibroblasts canbe stimulated to grow by a brief exposure to EDTA-trypsin, according totechniques well known to those skilled in the art. The exposure totrypsin is too brief to release the fibroblasts from their attachment tothe culture vessel wall. Immediately after the cultures have becomeestablished and are approaching confluence, samples of the fibroblastscan be processed for frozen storage, such as in liquid nitrogen.Presently, numerous methods for successfully freezing cells for lateruse are known in the art, and are included in the present invention. Thefrozen storage of early rather than late passage fibroblasts ispreferred because the number of passages in cell culture of normal humanfibroblasts is limited.

[0081] The fibroblasts can be frozen in any freezing medium suitable forpreserving fibroblasts. A medium consisting of about 70% (v/v) growthmedium, about 20% (v/v) fetal bovine serum and about 10% (v/v)dimethylsulfoxide (DMSO) can be used with good effect. DMSO can also besubstituted with, for example, glycerol. Thawed cells can be used toinitiate secondary cultures to obtain suspensions for use in the samesubject without the inconvenience of obtaining a second specimen.

[0082] Any tissue culture technique that is suitable for the propagationof dermal fibroblasts from biopsy specimens may be used to expand thecells to practice the invention. Techniques well known to those skilledin the art can be found in R. I. Freshney, Ed., ANIMAL CELL CULTURE: APRACTICAL APPROACH (IRL Press, Oxford, England, 1986) and R. I.Freshney, Ed., CULTURE OF ANIMAL CELLS: A MANUAL OF BASIC TECHNIQUES,Alan R. Liss & Co., New York, 1987), which are hereby incorporated byreference.

[0083] The medium can be any medium suited for the growth of primaryfibroblast cultures. The medium can be supplemented with human ornon-human serum in an amount of between about 0.0% and about 20% (v/v)to promote growth of the fibroblasts. Higher concentrations of serumpromote faster growth of the fibroblasts. An example of medium havingapplication herein comprises glucose DMEM supplemented with about 2 mMglutamine, about 10 mg/L sodium pyruvate, about 10% (v/v) fetal bovineserum and antibiotics (“complete medium”), wherein the concentration ofglucose ranges from about 1,000 milligrams per liter of medium to about4,500 milligrams per liter of medium. Fibroblasts can also be expandedin serum-free medium. Tissue culture growth medium used for culturingthe fibroblasts is generally supplemented with antibiotics to preventmicrobial (e.g., bacterial, fungal, yeast, and mycoplasma) contaminationof the cultures. Mycoplasma contamination is a frequent and particularlyvexatious problem in tissue culture. In order to prevent or minimizemycoplasma contamination, an agent such as tylosin can be added to therelevant culture medium. The medium can be further supplemented with oneor more (e.g., all) the following antibiotics: gentamicin,ciprofloxacine, alatrofloxacine, azithromycin, and tetracycline. Tylosincan be used at a concentration of 0.006-0.6 mg/ml (e.g., about 0.06)mg/ml. Gentamicin can be used at a concentration of 0.02-0.5 (e.g.,about 0.1) mg/ml. Ciprofloxacine can be used at concentration of0.002-0.05 (e.g., about 0.01) mg/ml. Alatrofloxacine can be used at aconcentration of 0.2-5.0 (e.g., about 1.0) μg/ml. Azithromycin can beused at a concentration of 0.002-0.05 (e.g., 0.01) mg/ml. Tetracyclinecan be used at a concentration of 0.004-0.1 (e.g., about 0.02) mg/ml.The antibiotics can be present for the whole period of the culture orfor only part of the culture period.

[0084] Mycoplasmal contamination was tested for by an agar culturemethod using a culture system such as, for example, a kit marketed bySigma, St. Louis, Mo., as “Mycoplasma Test Medium” (Cat. No. M 1914) andby PCR. PCR testing was performed by the both the ATCC (Manassas, Va.)and Esoterics Co., Houston, Tex. The ATCC markets a PCR mycoplasma testkit (“Mycoplasma Detection Kit”; cat. no. 90-100K). Using both the agarculture method and the PCR test, a low level of mycoplasmalcontamination was detected in four out of fifteen cultures performed inmedium supplemented with gentamicin as the only antibiotic; on the otherhand, no mycoplasmal contamination was detected in any of 12 culturescontaining tylosin (0.06 mg/ml), gentamicin (0.1 mg/ml), ciprofloxacine(0.01 mg/ml), alatrofloxacine (1.0 μg/ml), azithromycin (0.01 mg/ml),and tetracycline (0.02 mg/ml). The antibiotic mixture was present in thefibroblast cultures only for the first two weeks after initiation. Aftertwo weeks of culture antibiotic containing medium was replaced withantibiotic-free medium. Once sufficient cells had grown, they weretested for mycoplasmal as well as bacterial and fungal contamination.Only cells with no detectable contamination were used in the describedtreatment methods of the invention. Another agent that has been found bythe inventors to be useful in preventing mycoplasmal contamination is aderivative of 4-oxo-quinoline-3-carboxylic acid (OQCA) which is sold asMycoplasma Removal Agent (MRA) by ICN Pharmaceuticals, Inc. (Costa Mesa,Calif.) and was used according to the manufacturer's instructions. Thisderivative of OQCA can be used at a concentration of 0.1-2.5 (e.g., 0.5)μg/ml.

[0085] Autologous fibroblasts can be passaged into new flasks bytrypsinization. For expansion, individual flasks are split 1:3. Triplebottom, T-150 flasks, having a total culture area of 450 cm², aresuitable for the practice of the invention. A triple bottom T-150 flaskcan be seeded with about 1×10⁶ to about 3×10⁶ cells and has a capacityto yield about 8×10⁶ to about 1.0×10⁷ cells. When the capacity of theflask is reached, which typically requires about 5-7 days of culture,the growth medium is replaced by serum-free medium; thereafter the cellsare incubated, i.e., held at between bout 30° C. and about 37.5° C., forat least 4 hours (e.g., overnight or about 18 hours). The incubation ofthe cells in serum free medium substantially removes from the cellsproteins that are derived from the fetal bovine serum which, if present,can elicit an untoward immune response in the subject. In a preferredembodiment, serum-free medium comprises glucose DMEM supplemented withabout 2 mM glutamine, and about 110 mg/L sodium pyruvate, wherein theconcentration of glucose can range from approximately 1,000 mg/L ofmedium to about 4,500 mg/L of medium. In a preferred embodiment, theconcentration of glucose is approximately 4,500 mg/L of medium. Theserum-free medium can also contain the above-described antibiotics.

[0086] At the end of the incubation in serum free medium, the cells areremoved from the tissue culture flask by trypsin-EDTA; washedextensively by centrifugation and resuspension; and suspended forinjection in an equal volume of injectable isotonic solution with anappropriate physiological osmolarity, which is substantially pyrogen andforeign protein free. An example of such an isotonic solution isisotonic saline. Five triple bottom T-150 flasks, grown to capacity,yield about 3.5×10⁷ to about 7×10⁷ cells which are sufficient to make upabout 1.2 to about 1.4 ml of suspension. A pharmaceutically acceptablecarrier can then be added to the passaged autologous fibroblasts forminga pharmaceutical composition. The phrase “pharmaceutically acceptable”refers to molecular entities and compositions that are not deleteriousto the cells, are physiologically tolerable, and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human. Such compositionsinclude diluents of various buffer content (e.g., Tris-HCl, acetate,phosphate), pH and ionic strength.

[0087] Alternatively, the cells can be transported on ice at about 4° C.so long as they are injected within 24-48 hours of the time that thepharmaceutical composition is made. The cells can be suspended in anappropriate physiological solution with appropriate osmolarity andtested for pyrogen and endotoxin levels, except for the absence ofphenol red pH indicator, and the replacement of the fetal bovine serumby the subject's serum for such transportation (transport medium). Inanother embodiment the cells can be suspended in Krebs-Ringer solutioncomprising 5% dextrose or any other physiological solution. The cellscan be aspirated and injected in the transport medium.

[0088] The volume of saline or transport medium in which the cells aresuspended is related to such factors as the number of fibroblasts thepractitioner desires to inject, the extent of the damage due to tissuedegeneration or defect, or the size, the number of the defects that areto be treated, and the urgency of the subject's desire to obtain theresults of treatment. Moreover, the practitioner can suspend the cellsin a larger volume of medium and inject correspondingly fewer cells ateach injection site.

Device for Treating Degenerated Tissue with Autologous PassagedFibroblast Pharmaceutical Composition of the Present Invention

[0089] As explained above, the present invention extends to a devicedelivering a pharmaceutical composition of autologous passaged cellsdescribed above, to a point proximate to the site of tissuedegeneration, or defects of the palate, oral mucosa or skin. Such adevice comprises a hypodermic syringe having a syringe chamber, a pistondisposed therein, an orifice communicating with the chamber, apharmaceutical composition comprising autologous, passaged fibroblasts,and a pharmaceutically acceptable carrier thereof, such that thepharmaceutical composition is disposed in the chamber, and a hypodermicneedle is fixed to the orifice.

The Administration of a Pharmaceutical Composition

[0090] A pharmaceutical composition of the invention can be used totreat tissue degeneration in a subject as a result of a disorder ordisease, such as periodontal disease, or defects of the oral mucosa orskin, such as scars or wrinkles, by use of the following techniques.

[0091] Initially, the tissue to be injected is prepped with alcohol andstretched to give a taut surface. If the tissue degeneration is theresult of periodontal disease, the tissue to be injected is periodontaltissue, including periodontal pockets. If the tissue to be injectedcontains defects in the palate or gums of the subject, the tissue to beinjected with a pharmaceutical composition of the present invention issubadjacent to the defect. Also, if the tissue to be injected is theskin, in order to treat defects, e.g., scars, wrinkles, or any of thedefects listed above, it is also injected into the dermis orsubcutaneous tissue.

[0092] After the tissue to be injected has been prepped, a syringe isfilled with a pharmaceutical composition of the present invention andfitted with a 30 gauge needle. The needle is inserted into the tissue assuperficially as possible, and the orientation of the bevel is notcritical to the success of this method of the present invention. Theinjection of the pharmaceutical composition is made by gentle pressureuntil a slight blanch is seen in the injected tissue. Multiple serialinjections are made.

EXAMPLE II A Composition for Promoting the Regeneration of Tissue thathas Degenerated in a Subject

[0093] Also disclosed in the present invention is a composition forpromoting the regeneration of tissue that degenerated in a subject. Suchdegeneration can occur as a result of periodontal disease, trauma,dermatoses, recurrent aphthous stomatitis, or infections to name only afew. Other relevant diseases and disorders are listed above. Moreover,examples of periodontal disease which can cause tissue degenerationinclude periodontal degeneration, gingivitis, or non-healing wounds ofthe palatal mucosa or gingival mucosa. Moreover, a composition of thepresent invention can also be used to correct defects in tissue of asubject, such as, for example, defects in the palatal mucosa, gingivalmucosa, or defects in skin, e.g., any of those described above. Suchcompositions can be used, for example, for healing extraction socketsafter extraction of a tooth. Moreover, they can be used to fillextraction-associated bone defects and to rebuild bone in periodontaldisease. They can be used, for example, to repair dental ridges andmandibular bone, maxillary bone, and sinus floor defects. Indeed theycan be used to repair any bony defect in the body, including, forexample, non-union of fractured bones such as long bones. A compositionof the present invention comprises a biodegradable acellular matrix, andautologous passaged fibroblasts substantially free of immunogenicproteins (e.g., culture medium serum-derived proteins), wherein theautologous fibroblasts are integrated into the biodegradable acellularmatrix.

[0094] Moreover, disclosed herein is a method of making a composition ofthe present invention to promote regeneration of tissue in a subject.Such a method comprises providing a suspension of passaged autologousfibroblasts substantially free of immunogenic proteins, e.g., culturemedium serum-derived proteins, providing a biodegradable acellularmatrix, incubating the biodegradable acellular matrix with thesuspension of passaged autologous fibroblasts such that the autologousfibroblasts integrate within the biodegradable acellular matrix forminga composition for promoting regeneration of tissue. Also disclosedherein is a method of using a composition of the present invention,comprising identifying a site of tissue degeneration, and applying thecomposition for promoting regeneration of tissue to the site of tissuedegeneration.

[0095] In order to produce a composition of the present invention,autologous passaged fibroblasts substantially free of immunogenicproteins (e.g., culture medium serum-derived proteins) must be madeavailable to avoid the phenomenon of allograft rejection, which is wellknown to transplantation surgeons and immunologists. Hence, it isessential that the cultured fibroblasts be histocompatible with thehost. Histocompatibility can be ensured by obtaining a biopsy of thegums, palate or skin of the subject to be treated, and culturing thefibroblasts from this specimen.

[0096] Before the initiation of the dermal fibroblast culture, a biopsyof 1-3 mm is taken from the gums, palate or skin of the subject, andwashed repeatedly with antibiotic and antifungal agents (see above). Thespecimen of dermis is then separated into small pieces. The pieces ofthe specimen are individually placed onto a dry surface of a tissueculture flask and allowed to attach for between 5 and 10 minutes beforea small amount of medium is slowly added, taking care not to displacethe attached tissue fragments. After 48 hours of incubation, the flaskis fed with additional medium. When a T-25 flask is used to start theculture, the initial amount of medium is 1.5-2.0 ml. The establishmentof a cell line from the biopsy specimen ordinarily takes between 2 and 3weeks, at which time the cells can be removed from the initial culturevessel for expansion.

[0097] During the early stages of the culture it is desirable that thetissue fragments remain attached to the culture vessel bottom; fragmentsthat detach should be reimplanted into new vessels. The autologousfibroblasts can be stimulated to grow by a brief exposure totrypsin-EDTA, according to techniques known to those skilled in the art.The exposure to trypsin is too brief to release the fibroblasts formtheir attachment to the culture vessel wall.

[0098] Immediately after the cultures have become established and areapproaching confluence, samples of the autologous fibroblasts can beprocessed for frozen storage in, for example, liquid nitrogen. Thefrozen storage of early rather than late passage fibroblasts ispreferred because the number of passages in cell culture of normal humanfibroblasts is limited.

[0099] The autologous fibroblasts can be frozen in any freezing mediumsuitable for preserving cells. A medium consisting of about 70% growthmedium, about 20% (v/v) fetal bovine serum and about 10% (v/v)dimethylsulfoxide (DMSO) can be used with good effect. DMSO can also besubstituted with, for example, glycerol. Thawed cells can be used toinitiate secondary cultures to obtain suspensions for use in the samesubject without the inconvenience of obtaining a second specimen.

[0100] Any tissue culture technique that is suitable for the propagationof dermal fibroblasts from biopsy specimens may be used to expand thecells to practice the invention. Techniques for propagation known tothose skilled in the art can be found in R. I. Freshney, Ed., ANIMALCELL CULTURE: A PRACTICAL APPROACH (IRL Press, Oxford England, 1986) andR. I. Freshney, Ed., CULTURE OF ANIMAL CELLS: A MANUAL OF BASICTECHNIQUES, Alan R. Liss & Co., New York, 1987), which are herebyincorporated by reference.

[0101] The medium can be any medium suited for the growth of primaryautologous fibroblast cultures. In most instances, the medium issupplemented with human or non-human serum in the amount of betweenabout 0.0% and about 20% (v/v) to promote growth of the autologousfibroblasts. Higher concentrations of serum promote faster growth of thefibroblasts. In a preferred embodiment the serum is fetal bovine serum,which is added to a final concentration of about 10% of medium.Moreover, the medium can be glucose DMEM supplemented with about 2 mMglutamine, about 110 mg/L sodium pyruvate, about 10% (v/v) fetal bovineserum and antibiotics (“complete medium”), wherein the concentration ofglucose ranges from approximately 1,000 mg/L of medium to approximately4,500 mg/L of medium. Preferably, the concentration of glucose in themedium is approximately 4,500 mg/L of medium. The medium will generallyalso contain antibiotics such those described above.

[0102] Autologous fibroblasts can be passaged into new flasks bytrypsinization. For expansion, individual flasks are split 1:3. Triplebottom, T-150 flasks, having a total culture area of 450 cm², aresuitable for the practice of the invention. A triple bottom T-150 flaskcan be seeded with about 1×10⁶ to about 3×10⁶ to about 3×10⁶ cells andhas a capacity to yield about 8×10⁶ to about 1.0×10⁷ cells. When thecapacity of the flask is reached, which typically requires 5-7 days ofculture, the growth medium is replaced by serum-free medium; thereafterthe cells are incubated, i.e., held at between about 30° C. and about37.5° C., for at least 4 hours (e.g., overnight, or about 18 hours). Theincubation of the cells in serum free medium substantially removes fromthe cells proteins that are derived from the fetal bovine serum which,if present, would elicit an immune subject. In a preferred embodiment,the serum-free medium comprises glucose and DMEM supplemented with about2 mM glutamine, about 110 mg/L sodium pyruvate, wherein theconcentration of glucose ranges from approximately 1,000 mg/L of mediumto approximately 4,500 mg/L of medium, and preferably is 4,500 mg/L ofmedium.

[0103] A biodegradable acellular matrix is then provided. Examples ofsuch matrices which can be used in the present invention include, butare not limited to, acellular matrices comprising exogenous proteins, ormatrices comprising biodegradable polymers.

[0104] Numerous biodegradable acellular matrices comprising exogenousproteins are presently available, and have ready applications in thepresent invention. An embodiment of such biodegradable acellularmatrices are matrices comprising any type of collagen, or any type ofcollagen with glycosaminoglycans (GAG) cross-linked with, for example,glutaraldehyde. Examples of collagen matrices having application in thepresent invention are absorbable collagen sponges made by the CalcitekCompany of Carlsbad, Calif. These collagen sponge dressings, sold underthe names “COLLATAPE®,” “COLLACOTE®,” and “COLLAPLUG®” are made fromcross-linked collagen extracted from bovine deep flexor (Achilles)tendon, and glycosaminoglycans (GAGS). These products are soft, pliable,nonfriable, and non-pyrogenic. In addition, more than 90% of thisproduct consists of open pores. An alternative biodegradable acellularmatrix can consist of collagen (e.g., bovine porcine collagen type I)formed into a thin membrane. Such a membrane is manufactured by theCalcitek Company and is marketed as BioMend™. Another such membranousmatrix manufactured by ED. GEISTLICH SÖHNE AG of Wolhusen, Switzerland,is made of porcine type I and type III collagen and is marketed asBio-Gide®. Bio-Gide® has a bilayer structure with one surface that isporous allowing the ingrowth of cells and a second surface that is denseand will prevent the ingrowth of fibrous tissue. Another biodegradableacellular matrix can be made from bone spongiosa formed into granules orblocks. This material consists of animal (e.g., human, non-humanprimate, bovine, sheep, pig, or goat) bone from which substantially allorganic material (e.g., proteins, lipids, nucleic acids, carbohydrates,and small organic molecules such as vitamins and non-protein hormones)have been removed. This type of matrix is referred to herein as ananorganic matrix. One such matrix, which is marketed as either Bio-Oss®spongiosa granules or Bio-Oss® blocks, is manufactured by ED. GEISTLICHSÖHNE AG. This company also manufactures a block-type matrix (Bio-Oss®collagen), also consisting of the anorganic bone, but containing inaddition approximately 10% by weight of collagen fibers. Otherbiodegradable acellular matrices having applications in the presentinvention can contain one or more of gelatin, polyglycolic acid, or catgut sutures, demineralized bone, hydroxyapatite, or mixtures of thesesubstances. For example, a matrix made from demineralized human bone andformed into small blocks is marketed as Dynagraft™ matrix by GenSciRegeneration Laboratories, Inc. Demineralized bone can be combined, forexample, with collagen to produce a matrix in the form of a sponge,block, or membrane. Synthetic polymers made from one or more monomerscan also be used to make the biodegradable acellular matrices of theinvention. The matrices can be made from one or more of such syntheticpolymers. The synthetic polymers can also be combined with any of theabove-mentioned substances to form matrices. Different polymers forminga single matrix can be in separate compartments or layers. For example,Gore-Tex, Inc. manufactures a porous biodegradable acellular matrix(GORE RESOLUT XT Regenerative Material) that is composed of a syntheticbioabsorbable glycolide and trimethylene carbonate copolymer fiber (intowhich cells can migrate) attached to an occlusive membrane that does notpermit ingrowth of cells and composed of a synthetic bioabsorbableglycolide and lactide copolymer.

[0105] After a biodegradable acellular matrix has been selected, aconcentrated suspension of autologous passaged fibroblasts is evenlydistributed on the surface of the matrix. Using a concentratedsuspension is necessary to avoid going beyond the capacity of the matrixto absorb the liquid suspension. For example, a typical distribution ofcell suspension using the GORE RESOLUT XT as the matrix comprisesapplying about 94 μl to about 125 μl of cell suspension having about2.0×10⁶ cells to about 4.0×10⁶ cells, per square centimeter of matrix.Cells are allowed to attach to the matrix without further addition ofmedia. In a preferred embodiment, incubation of the cells with thematrix occurs at about 37° C. for about 1-2 hours. After at least sixtyminutes of incubation, the cells attach to the matrix material.Histological analysis of such matrices after seeding and incubating forat least on hour showed even distribution of cells throughout thematrices. At this time, the culture vessels containing the cell-loadedmatrices are supplemented with additional growth medium. Cells are thencultured in the matrix for about 3 to 4 days. The cells are added to thematrix at high density (see above) so as to substantially fill the spacewithin the matrix available for cells. As a result, little to no cellproliferation occurs during this 3-4 day culture period. Methods toestablish the appropriate number of autologous passaged fibroblasts toadd to any given acellular biodegradable matrix would be known to thosein the art. Indeed, it is undesirable for significant cell proliferationto occur during this period because the dividing fibroblasts secreteenzymes (e.g., collagenase) that can degrade or, at least, partiallydegrade the matrices. The matrix with the cells is then washed at leastthree times, for 10 minutes per wash, with, e.g., phosphate bufferedsolution (PBS) to substantially remove immunogenic proteins, e.g.,culture medium serum-derived proteins, which can elicit an immuneresponse in the subject. Fresh PBS is used for each washing. The matrixis then incubated twice for at least one hour per incubation in freshPBS prior to use. After incubation, the matrix comprising autologousfibroblasts is placed on the area of tissue degeneration, or defect,such as, for example, the periodontal pockets, and secured so that itcan not be moved from the site.

[0106] In the case of sponge matrix (e.g. Collacote®), approximately1.5-2.0×10⁷ fibroblasts in approximately 1.5 ml of growth medium areseeded onto 2 cm×4 cm thin (approximately 2.5 mm to 3.0 mm thick)sponges. The sponge is then incubated at 37° C. for about 1-2 hourswithout further addition of medium to allow substantially all thefibroblasts to adhere to the matrix material. After cell adherence,additional growth medium is added to the composition of matrix andfibroblasts which is then incubated at 37° C. for 3-4 days (with dailychange of medium). As explained above, little to no cell proliferationoccurs during this 3-4 day culture. The composition is then removed fromgrowth medium containing FBS and washed repeatedly (at least 3 times)with FBS-free PBS. After each addition of PBS, the matrix is incubatedfor 10-20 minutes prior to discarding of the PBS. After the final wash,the composition is either applied directly to the area of the subjectrequiring tissue regeneration or is transferred to a shipping vialcontaining a physiological solution (e.g., Kreb's Ringer solution) andshipped (preferably overnight) to a practitioner (e.g., a dentist orphysician).

[0107] In the case of a membranous matrix (e.g. BioMend™), approximately3-8×10⁶ fibroblasts in approximately 100 μl of growth medium are seededonto the 15 mm×20 mm thin (approximately 0.5 to 1.0 mm thick) membranes.The membrane is then incubated at 37° C. for about 30-60 minutes withoutfurther addition of medium to allow substantially all the fibroblasts toadhere to the matrix material. After cell adherence, additional growthmedium is added to the composition of matrix and fibroblasts which isthen incubated at 37° C. for 2-3 days (with daily change of medium). Thecells were added to the matrix at high density (see above) so as tosubstantially fill the space within the matrix available for cells withthe same result described above. Washing of the composition and eitherimmediate use or shipping are as described above for the spongematrices.

[0108] In the case of a block matrix such as the above describedanorganic matrix (e.g., the Bio-Oss® block) or a demineralized bonematrix (e.g., the Dynagraft™ matrix), approximately 1.2-2.0×10⁷fibroblasts in approximately 100 μl to 150 μl of growth medium areseeded into 1 cm×1 cm×2 cm cubic blocks of matrix material. Cells areslowly seeded onto one face of the block face. Once the medium and cellshave been absorbed into the block, another face of the block is seededin a similar fashion. The procedure is repeated until all faces of theblock have been seeded and the block is fully saturated with medium.Care is taken to avoid adding excess medium and thereby causing leakingout of medium and cells from the block. The composition is thenincubated at 37° C. for about 60-120 minutes without further addition ofmedium to allow substantially all the fibroblasts to adhere to thematrix material. After cell adherence, additional growth medium is addedto the composition of matrix and fibroblasts which is then incubated at37° C. for 2-3 days (with daily change of medium). The cells were addedto the matrix at high density (see above) so as to substantially fillthe space within the matrix available for cells with the same resultdescribed above. Washing of the composition and either immediate use orshipping are as described above for the sponge matrices.

[0109] Compositions using Bio-Oss® collagen, RESOLUT, COLLACOTE®,Dynagraft™ as the matrix material have been used to heal extractionsockets of 19 patients after extraction of a tooth. In untreatedextraction sockets, the subject's fibroblasts migrate into the socketsapproximately 10-14 days after extraction of the tooth and thus thehealing process only begins at that time. However, by implanting thecompositions containing the patient's fibroblasts into the socketsimmediately after extraction of the tooth (e.g., within 1-3 hours of theextraction), the healing process is initiated immediately. Furthermore,by implanting the compositions, shrinkage of the socket (due to collapseof the socket walls), which is generally approximately 30% in untreatedsockets, is minimized. It is particularly desirable that socketshrinkage be minimized in cases in which it is proposed to implant adental prosthesis (e.g., a false tooth) at the site of the extraction ata later date.

[0110] The inventors have also found that Bio-Oss® collagen matricesloaded with autologous fibroblasts to be useful in the repair ofextraction-associated mandibular and maxillary dental ridge defects. Intwo patients whose dental ridges had been treated with suchcompositions, after being in place for 6-18 months, there was nodetectable deterioration of the matrix material and the structureresembled normal bone.

[0111] In all the compositions used for the above described procedures,fibroblasts obtained from 1 mm punch biopsies of the patients' gumtissues were used and the compositions were shipped to the dentistsperforming the procedures. The compositions were shaped by the dentistto fit into the extraction sockets. In general, the compositions aftershaping had an approximately cylindrical shape with a length ofapproximately 10 mm and a diameter of approximately 4 mm. At the time ofimplanting, fragments of the compositions were prepared for histologicalanalysis. In all cases, fibroblast colonization of and proliferationwithin the matrices was seen by light microscopy.

[0112] The above-mentioned advantages (i.e., facile healing andmaintenance of socket volume) were seen in all cases. Furthermore,infection of the tissues surrounding the socket, which is a frequentlyobserved sequela of procedures in which extraction sockets are nottreated, was not observed in any case. Compositions using Bio-Oss®collagen (2 patients) as the matrix had the advantage of persistence ofthe matrix material in the sockets longer than those compositions inwhich the matrix material consisted of collagen only and thus providedphysical support for the fibroblasts before full replacement of thematrix material with components endogenous to the patient, e.g., cellsand extracellular matrix components.

EXAMPLE III An Injectable Composition for Correcting Skin Defects andAugmenting Tissue in a Subject

[0113] As explained above, the present invention extends to aninjectable composition for correcting a defect in skin of a subject, oraugmenting tissue of a subject, wherein the injectable compositioncomprises passaged autologous fibroblasts substantially free ofimmunogenic proteins (e.g., culture medium serum-derived proteins), anda biodegradable, acellular injectable filler material. Examples of skindefects that can be treated with the present invention include scars,particularly facial scars resulting from trauma or acne, or wrinkles.Any of the other skin and other tissue defects listed above can also betreated with the present invention. Furthermore, the present inventioncan be used to augment tissue in a subject. For example, the injectablecomposition of the present invention can be injected into the lips of asubject in order to make the lips larger and fuller, or it can beinjected below laugh lines to eliminate or diminish them. Furthermore,the compositions can be injected subcutaneously to treat subcutaneousdefects that can have arisen from congenital or acquired effects.Fibroblasts are used in the present invention because they can bereadily obtained and expanded, and are a cell type normally presentbeneath the dermis. Fibroblasts taken from a biopsy of the gums, palate,or skin of the subject can be used in the present invention, and canreadily obtained using procedures set forth above.

[0114] Initially, a dermal fibroblast culture is initiated from a 1 to 5mm full thickness biopsy specimen of the gums, palate or skin of thesubject. Because of the phenomenon of allograft rejection, which is wellknown to transplantation surgeons and immunologists, it is essentialthat the cultured fibroblasts be histocompatible with the host.Histocompatibility can be ensured by obtaining a biopsy of the subjectto be treated and culturing the fibroblasts from this specimen.

[0115] Before the initiation of the culture, the biopsy is washedrepeatedly with antibiotic and antifungal agents (see above). Thespecimen of dermis is then separated into small pieces. The pieces ofthe specimen are individually placed onto the dry surface of a tissueculture flask and allowed to attach for between about 5 and about 10minutes, before a small amount of medium is slowly added, taking carenot to displace the attached tissue fragments. After about 48 hours ofincubation, the flask is fed with additional medium. When a T-25 flaskis used to start the culture, the initial amount of medium isabout1.5-2.0 ml. The establishment of a cell line from the biopsyspecimen ordinarily takes between about 2 and 3 weeks, at which time thecells can be removed from the initial culture vessel for expansion.

[0116] During the early stages of the culture, it is desirable that thetissue fragments remain attached to the culture vessel bottom; fragmentsthat detach should be reimplanted into new vessels. The fibroblasts canbe stimulated to grow by a brief exposure to EDTA-trypsin, according totechniques well known to those skilled in the art. The exposure totrypsin is too brief to release the fibroblasts from their attachment tothe culture vessel wall.

[0117] Immediately after the cultures have become established and areapproaching confluence, samples of the fibroblasts can be processed forfrozen storage, such as in liquid nitrogen. Presently, numerous methodsfor successfully freezing cells for later use are known in the art andare included in the present invention. The frozen storage of earlyrather than late passage fibroblasts is preferred because the number ofpassages in cell culture of normal human fibroblasts is limited.

[0118] The fibroblasts can be frozen in any freezing medium suitable forpreserving fibroblasts. A medium consisting of about 70% (v/v) growthmedium, about 20% (v/v) fetal bovine serum and about 10% (v/v)dimethylsulfoxide (DMSO) can be used with good effect. DMSO can also besubstituted with, for example, glycerol. Thawed cells can be used toinitiate secondary cultures to obtain suspensions for use in the samesubject without the inconvenience of obtaining a second specimen.

[0119] Any tissue culture technique that is suitable for the propagationof dermal fibroblasts from biopsy specimens may be used to expand thecells to practice the invention. Techniques well known to those skilledin the art can be found in R. I. Freshney, Ed., ANIMAL CELL CULTURE: APRACTICAL APPROACH (IRL Press, Oxford, England, 1986) and R. I.Freshney, Ed., CULTURE OF ANIMAL CELLS: A MANUAL OF BASIC TECHNIQUES,Alan R. Liss & Co., New York, 1987), which are hereby incorporated byreference.

[0120] The medium can be any medium suited for the growth of primaryfibroblast cultures. The medium can be supplemented with human ornon-human serum in an amount of between about 0.0% and about 20% (v/v)to promote growth of the fibroblasts. Higher concentrations of serumpromote faster growth of the fibroblasts. An example of medium havingapplication herein comprises glucose DMEM supplemented with about 2 mMglutamine, about 110 mg/L sodium pyruvate, about 10% (v/v) fetal bovineserum and antibiotics (“complete medium”), wherein the concentration ofglucose ranges from approximately 1,000 mg/L of medium to 4,500 mg/L ofmedium, and preferably is 4,500 mg/L. Fibroblasts can also be expandedin serum-free medium (see above). Serum-containing and serum-free mediawill generally also contain one or more antibiotics such as those listedabove.

[0121] Autologous fibroblasts can be passaged into new flasks bytrypsinization. For expansion, individual flasks are split 1:3. Triplebottom, T-150 flasks, having a total culture area of 450 cm² aresuitable for the practice of the invention. A triple bottom T-150 flaskcan be seeded with about 1×10⁶ to about 3×10⁶ cells and has a capacityto yield about 8×10⁶ to about 1.0×10⁷ cells. When the capacity of theflask is reached, which typically requires about 5-7 days of culture,the growth medium is replaced by serum-free medium; thereafter the cellsare incubated, i.e., held at between about 30° C. and about 37.5° C.,for at least 2 hours. The incubation of the cells in serum-free mediumsubstantially removes from the cells proteins that are derived from thexenogeneic (e.g., fetal bovine) serum which, if present, can elicit animmune response in the subject. In a preferred embodiment, serum-freemedium comprises glucose DMEM supplemented with about 2 mM glutamine,and about 110 mg/L sodium pyruvate, wherein the concentration of glucoseis approximately 1,000 mg/L of medium to approximately 4,500 mg/L ofmedium, and preferably 4,500 mg/L of medium.

[0122] At the end of the incubation in serum-free medium, the cells areremoved from the tissue culture flask by trypsin-EDTA; washedextensively by centrifugation and resuspension; and suspended forsubsequent use in an injectable composition of the present invention, orfor injection into the subject. Five triple bottom T-150 flasks, grownto capacity, yield about 8×10⁶ to about 1.0×10⁷ cells per flask whichare sufficient to make up about 1.2 to 1.4 ml of cell suspension.

[0123] The cells can be transported at on ice 4° C. so long as they areinjected within 24-48 hours of their suspension. The cells can besuspended in an appropriate physiological solution with appropriateosmolarity and tested for pyrogens and endotoxin levels, except for theabsence of phenol red pH indicator, and the replacement of the fetalbovine serum by the subject's serum for such transportation (transportmedium). In another embodiment the cells can be suspended inKrebs-Ringer solution comprising 5% dextrose or any other physiologicalsolution. The cells can be aspirated and injected into the transportmedium.

[0124] The volume of saline or transport medium in which the cells aresuspended is related to such factors as the number of fibroblasts thepractitioner desires to inject, the extent of the defects to thesubject's skin that are to be corrected, the size or number of thedefects that are to be corrected, and the urgency of the subject'sdesire to obtain the results of the treatment. Moreover, thepractitioner can suspend the cells in a larger volume of medium andinject correspondingly fewer cells at each injection site. In aninjectable composition of the present invention, the passaged autologousfibroblasts of the present invention are mixed with a biodegradable,acellular injectable filler material in a ratio of approximately 1:1 byvolume.

Biodegradable, Acellular Injectable Filler Material

[0125] As explained above, an injectable composition of the presentinvention also comprises a biodegradable, acellular injectable fillermaterial. Numerous types of biodegradable, acellular injectable fillermaterials are presently available and have applications in the presentinvention. More specifically, the filler material can be comprisedendogenous proteins, such as any type of collagen from the subject. Anexample of such a filler is “AUTOLOGEN” produced by Collagenesis, Inc.“AUTOLOGEN” is a dispersion of autologous dermal collagen fibers fromthe subject, and should not elicit an immune response. In order toobtain “AUTOLOGEN” for the subject, a specimen of tissue is obtainedfrom the subject and forwarded to Collagenesis, Inc., where it is turnedinto “AUTOLOGEN”. Approximately a one and a half square inch of tissueyields one cubic centimeter (cc) of “AUTOLOGEN”. After “AUTOLOGEN” hasbeen prepared, its concentration can be adjusted depending upon theamount needed to correct defects in the subject's skin, or augmenttissue in the subject. In particular, the concentration of “AUTOLOGEN”in the dispersion can be at least about 25 mg/L.

[0126] Another example of filler material comprises exogenous proteins,such as any type of collagen. Presently, numerous collagen products arecommercially available and have applications in the present invention.Examples of such products are reconstituted bovine collagen productscommercially available including, but not limited to, “ZYDERM I”,“ZYDERM II” and “ZYPLAST”, which comprise reconstituted bovine collagenfibers cross-linked with glutaraldehyde. These three products, whichhave been approved by the U.S. Food and Drug Administration (FDA) fortreating wrinkle lines and depressed scars since 1981, are produced bythe Collagen Corporation of Palo Alto, Calif.

[0127] Other examples of filler materials having applications in thepresent invention include, but are not limited to, solubilized gelatin,polyglycolic acid, or cat gut sutures. One particular is a gelatinmatrix implant sold under the mark “FIBRIL”, which comprises porcinegelatin powder plus o-aminocaproic acid, which are dispersed in a 0.9%(by volume) sodium chloride solution and an aliquot of plasma from thesubject, mixed in a 1:1 ratio by volume.

Methods for Correcting a Defect in Skin or Augmenting Tissue

[0128] Furthermore, the present invention extends to methods forcorrecting a defect in skin of a subject, such as scars, wrinkles or anyof the other skin, bone or subcutaneous defects, disorders, or diseaseslisted herein. It can be used for augmenting tissue in the subject,particularly facial tissue, e.g., augmenting the tissue of lips to makethe lips appear fuller. One such method of the present inventioncomprises injecting an effective amount of an injectable compositioncomprising autologous fibroblasts substantially free of immunogenicproteins (e.g., culture medium serum-derived proteins) and abiodegradable, acellular injectable filler material, into the subject atthe site of the skin defect or desired tissue augmentation so thatregeneration of tissue at the site is promoted.

[0129] Another method encompassed by the present invention comprises thesteps of injecting passaged autologous fibroblasts substantially free ofimmunogenic proteins (e.g., culture medium serum-derived proteins) intothe subject at a site of a skin defect or desired tissue augmentation,and injecting a biodegradable, acellular injectable filler material intothe site. Preferably, the duration between injecting the autologousfibroblasts into the subject and injecting the biodegradable acellularinjectable filler into the subject is about two weeks.

[0130] Passaged, autologous fibroblasts substantially free ofimmunogenic proteins (e.g., culture medium serum-derived proteins) canbe readily obtained using procedures set forth above. Furthermore, thevarious types of biodegradable, acellular filler materials, which havebeen described in detail above, have applications in the methodsdescribed herein.

[0131] Injections set forth in the present invention are typicallycarried out with a hypodermic syringe having a syringe chamber, a pistondisposed therein, an orifice communicating with the chamber, and ahypodermic needle is fixed to the orifice. The size of the needle usedin a method for correcting a defect in skin of a subject or augmentingtissue in the subject ranges from approximately 30 gauge toapproximately 27 gauge. With more viscous compositions needles of, e.g.,14 to 16 gauge can be used.

[0132] Initially, the tissue to be injected is prepped with alcohol andstretched to give a taut surface. After the tissue to be injected hasbeen prepped, a syringe is filled with an injectable composition of thepresent invention, if the first method described above is to be used, orwith passaged, autologous fibroblasts substantially free of immunogenicproteins (e.g., culture medium serum-derived proteins) if the secondmethod for correcting skin defects or augmenting tissue is used. Theneedle is inserted into the tissue as superficially as possible, and theorientation of the bevel is not critical to the success of this methodof present invention. The actual injection is made by gentle pressureuntil a slight blanch is seen in the injected tissue. Multiple serialinjections can be made. Approximately two weeks after passagedautologous fibroblasts are injected into the subject's skin, an equalvolume of biodegradable, acellular filler is injected using the sameprocedure as described above into the same location where the passagedautologous fibroblasts were previously injected.

[0133] The present invention is not to be limited in scope by thespecific embodiments described above, which are intended asillustrations of aspects of the invention. Here functionally equivalentmethods and components are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein, will become apparent to those skilled in the art fromthe foregoing description. Such modifications are intended to fallwithin the scope of the appended claims. Moreover, all cited referencesare, hereby, incorporated by reference.

What is claimed is:
 1. A composition for repairing tissue that hasdegenerated in a subject as a result of a disease, disorder, or defectin said subject, wherein said composition comprises: a biodegradableacellular matrix; and autologous fibroblasts, wherein the composition issubstantially free of culture medium serum-derived proteins, and whereinsaid autologous fibroblasts are integrated within said acellular matrix.2. The composition of claim 1, wherein said biodegradable acellularmatrix comprises exogenous proteins.
 3. The composition of claim 2,wherein said biodegradable acellular matrix comprises any type ofcollagen.
 4. The composition of claim 2, wherein said biodegradableacellular matrix comprises any type of collagen and glycosaminoglycans,cross-linked with glutaraldehyde.
 5. The composition of claim 1, whereinsaid biodegradable acellular matrix comprises one or more substancesselected from the group consisting of gelatin, polyglycolic acid, catgut, demineralized bone, and hydroxyapatite.
 6. The composition of claim5, wherein said biodegradable acellular matrix comprises gelatin,polyglycolic acid, or cat gut.
 7. The composition of claim 1, whereinsaid disease, disorder, or defect comprises a defect of an oral mucosa,trauma to an oral mucosa, periodontal disease, diabetes, a cutaneousulcer, venous stasis, a scar of skin, or a wrinkle of skin.
 8. Thecomposition of claim 1, wherein said disease, disorder, or defect isperiodontal disease, and said periodontal disease comprises periodontaldegeneration, gingivitis, or non-healing wounds of a palatal mucosa or agingival mucosa.
 9. The composition of claim 1, wherein said autologousfibroblasts are from gums, palate, or skin of said subject.
 10. A methodfor making a composition for repairing tissue that has degenerated in asubject as a result of a disease, disorder, or defect in said subject,wherein said method comprises: providing a suspension of autologous,passaged fibroblasts; providing a biodegradable acellular matrix;incubating said suspension of autologous passaged fibroblasts with saidbiodegradable acellular matrix such that said autologous passagedfibroblasts integrate within said biodegradable acellular matrix,wherein sufficient autologous, passaged fibroblasts integrate withinsaid biodegradable acellular matrix to substantially fill the space onand within said biodegradable acellular matrix available for cells, andremoving substantially all culture medium serum-derived proteins fromsaid biodegradable acellular matrix and said integrated fibroblasts toform a composition for repairing tissue.
 11. The method of claim 10,wherein said biodegradable acellular matrix comprises exogenousproteins.
 12. The method of claim 11, wherein said acellular matrixcomprises any type of collagen.
 13. The method of claim 12, wherein saidacellular matrix comprises any type of collagen and glycosaminoglycans,cross-linked with glutaraldehyde.
 14. The method of claim 9, whereinsaid biodegradable acellular matrix comprises one or more substancesselected from the group consisting of gelatin, polyglycolic acid, catgut, demineralized bone, and hydroxyapatite.
 15. The method of claim 14,wherein said biodegradable acellular matrix comprises gelatin,polyglycolic acid, or cat gut.
 16. The method of claim 10, wherein saiddisease, disorder, or defect comprises a defect of an oral mucosa,trauma to an oral mucosa, periodontal disease, diabetes, a cutaneousulcer, venous stasis, a scar of skin, or a wrinkle of skin.
 17. Themethod of claim 10, wherein said disease or disorder is periodontaldisease, and said periodontal disease comprises periodontaldegeneration, gingivitis, or a non-healing wound of a palatal mucosa ora gingival mucosa.
 18. The method of claim 10, wherein the step ofproviding a suspension of autologous, passaged fibroblasts substantiallyfree of immunogenic proteins comprises: collecting a biopsy of dermis ofsaid subject; separating dermal autologous fibroblasts from said biopsy;culturing said dermal autologous fibroblasts in a culture mediumcomprising (a) between 0.0% and about 20% human or non-human serum and(b) a reagent that prevents the growth of mycoplasma; and exposing saidincubated dermal autologous fibroblasts to a proteolytic enzyme so as tosuspend fibroblasts.
 19. The method of claim 18, wherein the step ofcollecting a biopsy of dermis further comprises collecting a biopsy fromgums, palate or skin of said subject.
 20. A method of repairing tissuein a subject, wherein said method comprises: a) providing a compositioncomprising of autologous, passaged fibroblasts integrated into abiodegradable acellular matrix; b) identifying a site in said subject of(i) tissue degeneration due to a disease or disorder or (ii) a tissuedefect; c) placing the composition on the site so that said tissuedegeneration or said tissue defect is repaired.
 21. A method forrepairing tissue that has been damaged in a subject, wherein the methodcomprises: a) providing a pharmaceutical composition comprisingautologous passaged fibroblasts substantially free of immunogenicproteins, and a pharmaceutically acceptable carrier thereof; b)identifying a site in said subject of (i) tissue degeneration due to adisease or disorder or (ii) a tissue defect; c) injecting atherapeutically effective amount of the pharmaceutical compositionsubadjacent to the tissue degeneration or defect until a slight blanchis seen in the injected tissue.
 22. The method of claim 21, wherein saidtissue degeneration or said tissue defect in said subject comprisesdefects of an oral mucosa, trauma to an oral mucosa, periodontaldisease, diabetes, cutaneous ulcers, venous stasis, scars of skin, orwrinkles of skin.
 23. The method of claim 22, wherein said tissuedegeneration or said tissue defect is periodontal disease, wherein saidperiodontal disease comprises periodontal degeneration, gingivitis, or anon-healing wound of a palatal mucosa or a gingival mucosa.
 24. Themethod of claim 21, wherein the step of providing a pharmaceuticalcomposition comprises: a) taking a biopsy of dermis comprisingfibroblasts from a subject; b) separating fibroblasts from said biopsyso as to provide fibroblasts substantially free of extracellular matrixand non-fibroblast cells; c) placing the fibroblasts in a culture mediumcomprising between 0.0% and about 20% serum in order to growfibroblasts; d) incubating the fibroblasts in a serum-free medium for atleast 2 hours at between about 30° C. and about 37.5° C. to formpassaged fibroblasts; e) exposing the passaged fibroblasts to aproteolytic enzyme so as to suspend the passaged fibroblasts; and f)adding a pharmaceutically acceptable carrier to the suspended passagedfibroblasts to form the pharmaceutical composition.
 25. The method ofclaim 24, wherein biopsy is taken from gums, palate, or skin of thesubject.
 26. The method of claim 24 wherein said proteolytic enzyme istrypsin.
 27. A pharmaceutical composition for promoting regeneration oftissue that has been damaged in a subject comprising passaged autologousfibroblasts substantially free of culture medium serum-derived proteins,and a pharmaceutically acceptable carrier thereof.
 28. Thepharmaceutical composition of claim 27, wherein said passaged autologousfibroblasts comprise fibroblasts of the gums, palate, or skin.
 29. Aninjectable composition for correcting a defect in skin of a subject, oraugmenting tissue of a subject, said injectable composition comprising:a) autologous fibroblasts substantially free of culture mediumserum-derived proteins; and b) a biodegradable, acellular injectablefiller material.
 30. The injectable composition of claim 29, whereinsaid autologous fibroblasts are from gums, palate of skin of saidsubject.
 31. The injectable composition of claim 29, wherein saidbiodegradable, acellular injectable filler material comprises endogenousproteins.
 32. The injectable composition of claim 31, wherein saidbiodegradable acellular injectable filler material comprises aninjectable dispersion of autologous collagen fibers.
 33. The injectablecomposition of claim 32, wherein the concentration of autologouscollagen fibers in said injectable dispersion is at least 24 mg/ml. 34.The injectable composition of claim 29, wherein said biodegradableacellular injectable filler material comprises exogenous proteins. 35.The injectable composition of claim 34, wherein said exogenous proteinscomprise any type of collagen.
 36. The injectable composition of claim35, wherein said biodegradable, acellular injectable filler materialcomprises reconstituted bovine collagen fibers cross-linked withglutaraldehyde.
 37. The injectable composition of claim 29, wherein saidbiodegradable, acellular injectable filler material comprisessolubilized gelatin, polyglycolic acid, or cat gut.
 38. The injectablecomposition of claim 37, wherein said biodegradable, acellularinjectable filler material comprises porcine gelatin powder and aminocaproic acid dispersed in sodium chloride solution and an aliquot ofplasma from said subject.
 39. The injectable composition of claim 38,wherein the ratio of said sodium chloride solution and said aliquot ofserum is 1:1 by volume.
 40. The injectable composition of claim 39,wherein said sodium chloride solution comprises 0.9% sodium chloride byvolume.
 41. A method for correcting a defect in skin of a subject, oraugmenting tissue of a subject, the method comprising injecting aneffective amount of an injectable composition comprising autologousfibroblasts substantially free of culture medium serum-derived proteinsand a biodegradable, acellular injectable filler material, into thesubject at the site of the skin defect or desired tissue augmentation sothat regeneration of tissue at the site is promoted.
 42. A method forcorrecting a defect in skin of a subject, or augmenting tissue of asubject, the method comprising the steps of: a) injecting autologousfibroblasts substantially free of immunogenic proteins into the subjectat a site of a skin defect or desired skin augmentation; and b)injecting a biodegradable, acellular injectable filler material into thesite.
 43. The method for correcting a defect in skin of a subject, oraugmenting tissue of a subject of claim 42, wherein the duration betweeninjecting the autologous fibroblasts into the subject and injecting thebiodegradable acellular injectable filler into the subject is about twoweeks.
 44. The method of claim 42, wherein the autologous fibroblastsare from gums, palate of skin of the subject.
 45. The method of claim42, wherein the biodegradable, acellular injectable filler materialcomprises endogenous proteins.
 46. The method of claim 45, wherein thebiodegradable, acellular injectable filler material comprises aninjectable dispersion of autologous collagen fibers.
 47. The method ofclaim 46, wherein the concentration of autologous collagen fibers in theinjectable dispersion is at least 24 mg/ml.
 48. The method of claim 42,wherein the biodegradable, acellular injectable filler materialcomprises exogenous proteins.
 49. The method of claim 48, wherein theexogenous proteins comprise any type of collagen.
 50. The method ofclaim 49, wherein the biodegradable, acellular injectable fillermaterial comprises reconstituted bovine collagen fibers cross-linkedwith glutaraldehyde.
 51. The method of claim 42, wherein thebiodegradable, acellular injectable filler material comprisessolubilized gelatin, polyglycolic acid, or cat gut.
 52. The method ofclaim 51, wherein the biodegradable, acellular injectable fillermaterial comprises porcine gelatin powder and aminocaproic aciddispersed in sodium chloride solution, and an aliquot of plasma from thesubject.
 53. The method of claim 52, wherein the ratio of sodiumchloride solution to the aliquot of serum is 1:1 by volume.
 54. Themethod of claim 53, wherein the sodium chloride solution comprises 0.9%sodium chloride by volume.
 55. The method of claim 42, wherein the ratioof autologous fibroblasts substantially free of immunogenic proteins tobiodegradable, acellular injectable filler material is approximately 1:1by volume.
 56. The composition of claim 1, wherein said biodegradableacellular matrix comprises anorganic bone.
 57. The composition of claim56, further comprising collagen.
 58. The method of claim 10, whereinsaid biodegradable acellular matrix comprises anorganic bone.
 59. Themethod of claim 58, said biodegradable acellular matrix furthercomprising collagen.
 60. The method of claim 20, wherein saidbiodegradable acellular matrix comprises exogenous proteins.
 61. Themethod of claim 60, wherein said biodegradable acellular matrixcomprises any type of collagen.
 62. The method of claim 60, wherein saidbiodegradable acellular matrix comprises any type of collagen andglycosaminoglycans, cross-linked with glutaraldehyde.
 63. The method ofclaim 20, wherein said biodegradable acellular matrix comprises one ormore substances selected from the group consisting of gelatin,polyglycolic acid, cat gut sutures, demineralized bone, andhydroxyapatite.
 64. The method of claim 20, wherein said disease,disorder, or defect comprises a defect of an oral mucosa, trauma to anoral mucosa, periodontal disease, diabetes, a cutaneous ulcer, venousstasis, a scar of skin, or a wrinkle of skin.
 65. The method of claim20, wherein said disease, disorder, or defect is periodontal disease,and said periodontal disease comprises periodontal degeneration,gingivitis, or non-healing wounds of a palatal mucosa or a gingivalmucosa.
 66. The method of claim 20, wherein said autologous fibroblastsare from gums, palate, or skin of said subject.
 67. The method of claim20, wherein said biodegradable acellular matrix comprises anorganicbone.
 68. The method of claim 67, said biodegradable acellular matrixfurther comprising collagen.
 69. The method of claim 64, wherein saidtrauma to the oral mucosa is an extraction socket resulting from anextraction of a tooth.
 70. The composition of claim 7, wherein saidtrauma to the oral mucosa is an extraction socket resulting from anextraction of a tooth.
 71. The method of claim 16, wherein said traumato the oral mucosa is an extraction socket resulting from an extractionof a tooth.
 72. The method of claim 18, wherein said reagent comprisestylosin.
 73. The method of claim 72, wherein said reagent furthercomprises one or more compounds selected from the group consisting ofgentamicin, ciprofloxacine, alatrofloxacine, azithromycin, andtetracycline.
 74. The method of claim 20, wherein said tissuedegeneration or said tissue defect is selected from the group consistingof a laugh line, a stretch mark, an acne scar, and subcutaneous atrophy.75. The method of claim 20, wherein said tissue degeneration or saidtissue defect is hypoplasia of a lip or a lip fold.
 76. The method ofclaim 20, wherein said tissue is facial bone.
 77. The composition ofclaim 1, wherein said tissue is facial bone.